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. 2012 May;61(5):1153-9.
doi: 10.2337/db11-1271. Epub 2012 Jan 20.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) enhances engraftment and angiogenesis of mesenchymal stem cells in diabetic hindlimb ischemia

Debin Lu  1 , Ling Zhang, Haihui Wang, Yan Zhang, Jian Liu, Jing Xu, Ziwen Liang, Wuquan Deng, Youzhao Jiang, Qinan Wu, Shufa Li, Zhihua Ai, Yuxu Zhong, Ying Ying, Hongyan Liu, Feng Gao, Zhonghui Zhang, Bing Chen
Affiliations

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) enhances engraftment and angiogenesis of mesenchymal stem cells in diabetic hindlimb ischemia

Debin Lu et al. Diabetes. 2012 May.

Abstract

To examine whether the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a key regulator linking angiogenesis and metabolism, could enhance the engraftment and angiogenesis of mesenchymal stem cells (MSCs) in diabetic hindlimb ischemia, we engineered the overexpression of PGC-1α within MSCs using an adenoviral vector encoding green fluorescent protein and PGC-1α, and then tested the survivability and angiogenesis of MSCs in vitro and in vivo. Under the condition of hypoxia concomitant with serum deprivation, the overexpression of PGC-1α in MSCs resulted in a higher expression level of hypoxia-inducible factor-1α (Hif-1α), a greater ratio of B-cell lymphoma leukemia-2 (Bcl-2)/Bcl-2-associated X protein (Bax), and a lower level of caspase 3 compared with the controls, followed by an increased survival rate and an elevated expression level of several proangiogenic factors. In vivo, the MSCs modified with PGC-1α could significantly increase the blood perfusion and capillary density of ischemic hindlimb of the diabetic rats, which was correlated to an improved survivability of MSCs and an increased level of several proangiogenic factors secreted by MSCs. We identified for the first time that PGC-1α could enhance the engraftment and angiogenesis of MSCs in diabetic hindlimb ischemia.

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Figures

FIG. 1.
FIG. 1.
Optimization of adenovirus transfection efficiency by MSCs. A: Transfection efficiency of MSCs by Ad-GFP-PGC-1α. The MSCs were transfected with Ad-GFP-PGC-1α at different MOI and cultured for 48 h. The transfection efficiency was determined by flow cytometry. B: The morphology of rat MSCs and MSCs transfected with Ad-GFP-PGC-1α (defined as PGC-1α-MSC) or Ad-GFP (defined as GFP-MSC) at an MOI of 100 for 48 h was observed by fluorescence microscope (original magnification ×200). (A high-quality color representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
Proangiogenic factor expression of MSCs modified with genes in vitro. A: Western blotting analysis for Hif-1α, PGC-1α, and GAPDH. The protein expression of Hif-1α was not detected in MSCs and MSCs transfected with Ad-GFP (defined as GFP-MSC), but could be detected in MSCs transfected with Ad-GFP-PGC-1α (defined as PGC-1α-MSC) under 6 h normal culture. And after 6 h hypoxia and serum deprivation–conditioned culture, PGC-1α-MSCs produced more Hif-1α by 2.8-fold (compared with MSCs; P < 0.001) or by 3.0-fold (compared with GFP-MSCs; P < 0.001). B: Levels of VEGF secreted by MSCs, GFP-MSCs, and PGC-1α-MSCs from 24 to 72 h normal culture. C: Levels of PDGF-B secreted by MSCs, GFP-MSCs, and PGC-1α-MSCs from 24 to 72 h normal culture. D: Levels of FGF-2 secreted by MSCs, GFP-MSCs, and PGC-1α-MSCs from 24 to 72 h normal culture. *P < 0.01, PGC-1α-MSC vs. MSC; #P < 0.01, PGC-1α-MSC vs. GFP-MSC.
FIG. 3.
FIG. 3.
Apoptosis analysis of MSCs modified with genes in vitro. A: Three representative density plots of flow cytometry showed that the survival rate in the PGC-1α-MSC group was 61.8%, 35.8% in the GFP-MSC group, and 38.5% in the MSC group after 12 h hypoxia and serum deprivation culture. B: Survival rate in each group after 0, 6, and 12 h hypoxia and serum deprivation culture. *P < 0.01, PGC-1α-MSC vs. MSC; #P < 0.01, PGC-1α-MSC vs. GFP-MSC. C: Western blotting analysis for caspase 3, procaspase 3, β-actin, Bax, Bcl-2, and GAPDH after 12 h hypoxia and serum deprivation culture. (A high-quality color representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Perfusion recovery, capillary density, and necrosis incidence of ischemic hindlimb after stem cell–based therapy. A: Representative images of LDPI on 0, 7, and 14 days after therapy. The blood perfusion of ischemic hindlimb was markedly increased in the PGC-1α-MSC group. B: Representative examples of ischemic hindlimb muscles by alkaline phosphatase staining (original magnification ×100). C: Quantitative analysis of hindlimb blood perfusion. The LDPI index was significantly highest in the PGC-1α-MSC group on 7 and 14 days after transplantation, followed by the MSC and GFP-MSC groups, and the lowest was observed in the PBS group. D: Quantitative analysis of capillary density in ischemic hindlimb muscles. Capillary density was shown as capillary/muscle fiber ratio. The capillary/muscle fiber ratio of ischemic hindlimb muscles was highest in the PGC-1α-MSC group, followed by the MSC, GFP-MSC, and PBS groups. E: Incidence of limb necrosis 2 weeks after transplantation. Data in C and D were presented as mean ± S.E.M. &P < 0.01, PGC-1α-MSC group vs. PBS group; *P < 0.05, PGC-1α-MSC group vs. GFP-MSC group; #P < 0.05, PGC-1α-MSC group vs. MSC group; -P < 0.05, PBS group vs. GFP-MSC group; +P < 0.05, PBS group vs. MSC group. (A high-quality color representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Survival of MSCs modified with genes and secretion of VEGF and FGF-2 by transplanted MSCs in diabetic ischemic hindlimb. A: The GFP-positive MSCs (per 1,000 nuclei) in ischemic muscles of the PGC-1α-MSC group 5 days after transplantation were significantly higher than those in the GFP-MSC group. *P < 0.001. B: Western blotting analysis for VEGF, FGF-2, and GAPDH 5 days after transplantation. C: Secretion of FGF-2 and VEGF by transplanted MSCs that were modified with genes. Colocalization of GFP-positive MSCs (green) with VEGF (red) or GFP-positive MSCs (green) with FGF-2 (red) in ischemic muscles 5 days after transplantation. Scale bars, 10 μm. (A high-quality color representation of this figure is available in the online issue.)
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